Patterns of resistance to exonuclease digestion of oligonucleotides containing polycyclic aromatic hydrocarbon diol epoxide adducts at N6 of deoxyadenosine.

The effect of adduct stereochemistry on the susceptibility to hydrolysis by snake venom (VPD) and bovine spleen (SPD) phosphodiesterases was investigated with short deoxyoligonucleotides containing defined adducts derived from alkylation of the exocyclic 6-amino group of dA by polycyclic aromatic hydrocarbon diol epoxides (DEs). In accordance with several earlier reports, we have found that adducts with R configuration at the site of attachment of dA to the DE moiety derived from either benzo[a]pyrene (BaP) or benzo[c]phenanthrene (BcPh) are generally more resistant to hydrolysis by VPD than are their (S)-diastereomers. The reaction with VPD initially yields a fragment containing the adducted dA residue at its 3'-end, which slowly hydrolyzes to a dimer (pXpA*) with an intact 5'-phosphodiester bond to the adducted dA. With several of the adducts studied, this dimer underwent cleavage to release eventually the monomeric adduct p(dA*). Adducts derived from cis opening of the epoxide ring of both BaP and BcPh DEs were considerably more resistant to VPD than the corresponding trans-opened adducts. Although several previous investigations had suggested that oligonucleotides containing adducts which have S configuration at the site of attachment of the hydrocarbon to adenine are more resistant to cleavage by SPD than are their (R)-diastereomers, the present results with a more extensive set of oligonucleotides indicate that SPD, in contrast to VPD, exhibits little discrimination between adducts with R and S configuration at the site of attachment to the base. Notably, for both enzymes, the most resistant internucleotide linkage (the bond 3'-sugar to phosphate for VPD and 5'-sugar to phosphate for SPD) is between the modified base and the base immediately 5' to it, regardless of the configuration of the adduct.

Influence of adduct position and sequence length on the ligation of oligonucleotides containing benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts into M13mp7L2.

The adduct that would arise from cis opening of (+)-(1S,2R,3R, 4S)-3,4-dihydroxy-1,2-epoxy-benzo[c]phenan-threne (benzo[c]phenanthrene diol epoxide-2, where the benzylic hydroxyl group and the epoxide oxygen are trans) by the exocyclic N6-amino group of deoxyadenosine was incorporated at the marked site into four oligonucleotides, 5'-CAGA*TTTAGAGTCTGC-3', 5'-CAGTGCAGA*TTTAGAG-3', 5'-GTGCAGA*TTTAGA-3' and 5'-TGCAGA*TTTA-3'. The oligonucleotides were inserted into M13mp7L2 and the vector transfected into SOS-induced Escherichia coli SMH77 which were then plated on agar plates. The experiments reported here were designed to test the effect of the lesion position (the marked A in the sequences above) on the ligation efficiency of the insert and the frequency of failed constructs, as well as any possible effects on the mutagenic consequences of the lesion. The construct survival was estimated from the number of plaques formed following transformation, and mutation frequencies were estimated from sequencing of randomly picked plaques. Moving the adduct site to the middle of the sequence increased considerably the ligation efficiency regardless of the length of the inserted oligonucleotide, and changing the insert length or the adduct location did not markedly affect the frequency (40-58.6%) or distribution of mutations observed. Thus, so long as the local sequence (five or six bases surrounding the adduct) remains constant, the size of the oligonucleotide insert and the position of the adduct in it can be adjusted to give optimal ligation efficiency without altering the mutagenic consequences of the lesion.

Factors determining mutagenic potential for individual cis and trans opened benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts.

Four adducts that would result from trans opening at C-1 of benzo[c]phenanthrene 3,4-diol 1,2-epoxide (B[c]PhDE) isomers (i.e., DE-1 enantiomers, where the epoxide oxygen and benzylic hydroxyl group are cis, and DE-2 enantiomers, where they are trans) by the N(6)-amino group of dAdo, together with the two cis opened N(6)-dAdo adducts of B[c]PhDE-1, were incorporated into two oligonucleotides at the underlined site in 5'-TTTAGAGTCTGCTCCC [context I(A)] and 5'-CAGATTTAGAGTCTGC [context II(A)]. After ligation of these, and the corresponding unsubstituted oligonucleotides, into single-stranded M13mp7L2 bacteriophage and transfection into SOS-induced Escherichia coli SMH77, base substitution mutations induced by the different B[c]PhDE-dAdo adducts were determined. These findings were compared with data [Pontén et al. (1999) Biochemistry 38, 1144-1152] for cis opened B[c]PhDE-2-dAdo adducts in the same sequence contexts. In most cases, adducts with S absolute configuration at the site of attachment of the nucleoside to the hydrocarbon had higher mutation frequencies (1.9-56.5%) than the corresponding adducts with R configuration (0.05-5.6%). For adducts derived from B[c]PhDE-1, the predominant mutations were A-->T transversions in context I(A) and A-->G transitions for most of these adducts in context II(A). For adducts derived from B[c]PhDE-2, A-->T base substitutions predominated for most of the trans adducts, but A-->G mutations were favored by the cis adduct with S configuration in either context. Thus, the structural feature that most dramatically affected mutagenic activity was the configuration of the carbon at the attachment point, with S configuration mostly being associated with greater mutagenicity than the R configuration. However, other structural variations and sequence context also affected mutagenicity, indicating that a combination of structure and context effects define mutagenicity.

Mutational consequences of replication of M13mp7L2 constructs containing cis-opened benzo[a]pyrene 7,8-diol 9, 10-epoxide-deoxyadenosine adducts.

The four adducts that arise by cis ring opening of the four optically active benzo[a]pyrene diol epoxides by the exocyclic N6-amino group of deoxyadenosine were incorporated synthetically into each of two different oligonucleotide 16-mers, 5'-TTTXGAGTCTGCTCCC-3' [context I(A)] and 5'-CAGXTTTAGAGTCTGC-3' [context II(A)], at the X position. The eight resultant oligonucleotides were separately ligated into bacteriophage M13mp7L2 and replicated in Escherichia coli that had been SOS-induced, and the progeny were analyzed to evaluate the consequences of replication past these adducts. The presence of these adducts reduced plaque yields substantially. However, the progeny obtained exhibited high frequencies of base substitution mutation ranging from 9 to 68%, depending upon the individual adduct and the sequence context in which it was placed. For most of the adducts, A --> T transversion was the mutation found most frequently in either sequence context, and mutation frequencies in context I(A) were always substantially greater than those in context II(A). In context I(A), adducts with an R configuration at the site of nucleoside attachment were more mutagenic than those with an S configuration. In both sequence contexts that were studied, the cis adduct arising from the (7S,8R)-diol (9S,10R)-epoxide was the most mutagenic adduct. These findings clearly show that individual mutation frequencies are determined by the combined effects of both adduct structure and sequence context.

Sequence context effects on mutational properties of cis-opened benzo[c]phenanthrene diol epoxide-deoxyadenosine adducts in site-specific mutation studies.

Diastereomeric N6-substituted dAdo adducts (cis B[c]PhDE-2/1R and cis B[c]PhDE-2/1S) that correspond to cis-opening at C-1 of the enantiomeric benzo[c]phenanthrene 3,4-diol 1,2-epoxides in which the epoxide oxygen and the benzylic hydroxyl group are trans (DE-2) were synthetically incorporated into oligonucleotide 16-mers. Each adduct was placed at the fourth nucleotide from the 5'-end of each of two different oligonucleotide sequences derived from the E. coli supF gene. Each adduct was also placed in two additional oligonucleotide sequences that were constructed by interchanging the adduct site and the immediately adjacent nucleotides between the two original sequences. These oligonucleotides were designed for use in site-specific mutation studies, with a single-stranded bacteriophage M13mp7L2 vector, to determine if the effects of sequence context on types and frequencies of base substitution mutations are attributable only to nucleotides immediately adjacent to these polycyclic aromatic hydrocarbon diol epoxide-dAdo adducts, or whether more distant nucleotide residues also affect the mutagenic response. In SOS-induced Escherichia coli SMH77, total base substitution mutation frequencies for the cis B[c]PhDE-2/1R-dAdo adduct were relatively low (0.62-5.6%) compared with those for the cis B[c]PhDE-2/1S-dAdo adduct (11.9-56.5%). Depending on sequence context, cis B[c]PhDE-2/1R-dAdo gave predominantly A-->T or a more equal distribution of A-->T and A-->G mutations whereas cis B[c]PhDE-2/1S-dAdo gave either predominantly A-->T or predominantly A-->G base substitutions. Our results clearly indicate that nucleotides that are distal as well as those that are proximal to the adduct site are capable of influencing both the mutation frequency and the distribution of base substitution mutations.